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医药导报
医药导报, 2020, 39(12): 1626-1630
doi: 10.3870/j.issn.1004-0781.2020.12.006
多穗柯总黄酮对四氯化碳致小鼠急性肝损伤的保护作用
Protective Effect of Total Flavonoids from Lithocarpus Polystachyus Rehd. on CCl4-induced Acute Liver Injury in Mice
刘晓东, 温雯静, 赵志军,
漯河医学高等专科学校,河南漯河 462002
LIU Xiaodong, WEN Wenjing, ZHAO Zhijun,
Luohe Medical College, Luohe 462002,China
通信作者: 赵志军(1965-),男,河南巩义人,教授、主任医师,硕士生导师,从事临床超声研究。电话:0395-2112888,E-mail:lhyzzzj@163.com。

作者简介: 刘晓东(1978-),男,河南西平人,副教授,硕士,主要从事人体解剖学生物发育。ORCID:0000-0002-8367-8460,电话:0395-2964816,E-mail:604112525@qq.com。
基金资助: 河南省科技攻关计划资助项目(162102310596)
中图分类号: R963;R575.5     文献标识码: A     文章编号: 1004-0781(2020)12-1626-05
摘要:

目的 探讨多穗柯总黄酮(TFL)对四氯化碳(CCl4)致小鼠急性肝损伤的保护作用及其机制。方法 小鼠随机均分为6 组,正常对照组、模型对照组(6% CCl4橄榄油溶液)、水飞蓟素组(水飞蓟素120 mg·kg-1)、TFL低、中、高剂量组(50,100,150 mg·kg-1)。正常对照组、模型对照组灌胃等剂量0.9%氯化钠溶液,其余各组按剂量连续灌胃7 d,末次用药 2 h 后,按剂量5 mL·kg-1腹腔注射6% CCl4橄榄油溶液建立急性肝损伤模型,造模6 h后收集血清和肝脏组织。测定血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平以及肝组织白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)水平,苏木精-伊红(HE)染色进行常规组织学观察,Western blotting法检测肝组织中含NLR家族Pyrin域蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)、Caspase-1水平。结果 与模型对照组比较,多穗柯总黄酮可降低肝质量、肝指数(P<0.05),下调ALT、AST活力(P<0.05),降低肝组织 IL-1β、IL-6、TNF-α水平(P<0.05),增加肝组织SOD、GSH-Px水平,降低MDA水平(P<0.05),下调NLRP3、ASC、Caspase-1蛋白水平(P<0.05),镜下观察显示肝组织损伤得到不同程度改善。结论 多穗柯总黄酮对CCl4诱导的急性肝细胞损伤的保护作用,与减轻氧化应激水平,降低炎症因子水平,调控ROS/NLRP3/IL-1β信号通路有关。
关键词: 多穗柯总黄酮 ; 肝损伤 ; 急性 ; 抗炎 ; 抗氧化 ; NLRP3 炎性小体

Abstract:

Objective To investigate the protective effect and mechanism of total flavonoids from Lithocarpus polystachyus Rehd.(TFL) on CCl4-induced acute liver injury in mice. Methods Mice were randomly divided into 6 groups: normal control group, model control group (6% CCl4 olive oil solution), silymarin group (silymarin, 120 mg·kg-1), TFL low, medium and high dose group (50,100,150 mg·kg-1). The normal control group and the model control group were given equal volume of 0.9% of sodium chloride soution by gavage,while the other groups were administered with the corresponding dose of drugs according to the body mass. After 7 days, the acute liver injury model was established with 6% carbon tetrachloride olive oil solution (5 mL·kg-1). All of the mice were sacrificed to collect serum and liver tissues after 6 h. ALT and AST activities in serum were detected, and IL-1β, IL-6, TNF-α, SOD, GSH-Px, MDA levels in liver tissue were analyzed.Hematoxylin-eosin (HE) staining was used to observe the variation of liver histopathology.NLRP3, ASC, and Caspase-1 levelsin liver tissue were detected by Western blotting. Results Compared with the model control group, TFL decreased liver mass and liver index (P<0.05), down-regulated ALT and AST activities (P<0.05),decreased IL-1β, IL-6 and TNF-α levels in liver tissue (P<0.05), increased SOD and GSH levels, decreased MDA levels in liver tissue (P<0.05), and down-regulated NLRP3, ASC, and Caspase-1 protein levels in liver tissue (P<0.05). Microscopic observation showed that liver tissue injury was improved in different degrees. Conclusion The protective effect of TFL on CCl4-induced acuteliver injury was associated with attenuatedoxidative stress levels, reduced inflammatory factor levels, and regulated ROS/NLRP3/IL-1β signaling pathways.
Key words: Total flavonoids from ; Liver injury ; acute ; Anti-inflammatory ; Anti-oxidation ; NLRP3 inflammatory body

开放科学(资源服务)标识码(OSID):

肝脏是人体重要的代谢器官,肝损伤会对人的生活质量产生较大的影响。目前,伴随人们生活方式的改变以及环境的变化,肝病呈现明显的上升趋势。其中化学性肝损伤是比较常见的发病原因,严重时易导致肝硬化和肝癌的发生[1]。四氯化碳(CCl4)是常用的诱导肝损伤的化学溶剂,容易导致肝细胞分泌大量的炎症因子,加大炎症反应,同时还可通过促进细胞膜脂质过氧化,诱发肝损伤的发生[2]。

多穗柯(lithocarpus polystachyus Rehd.) 为常见的民间草药,具有清热利尿、滋润肝肾等功效。研究显示,多穗柯含有丰富的黄酮类化合物,含量达到10%,具有抗炎、抗氧化、降血脂等功能,但对于肝损伤的影响鲜有报道[3]。本研究采用CCl4诱导小鼠肝损伤建立动物模型,观察多穗柯总黄酮(total flavonoids extracts of Lithocarpus polystachyus Rehd,TFL)对其保护作用,并分析其保肝作用的机制。

1 材料与方法
1.1 动物

健康清洁级昆明小鼠60只,雄性,体质量(22±2) g,购自河南省实验动物中心,实验动物生产许可证号:SCXK(豫)2015-0005,使用许可证号:SYXK(豫)2016-0002。饲养温度为(24±2) ℃,12 h光照/12 h黑暗的室内,常规饲料,自由饮水进食,适应1 周后进行实验。

1.2 实验药物

多穗柯采集于贵州省贵阳市花溪区,经漯河医学高等专科学校赵喜兰副教授鉴定为壳斗科栎属常绿植物。按照预实验,准确秤取干燥的多穗柯叶5 kg,选择70%乙醇按照1:20 的料液浸泡12 h,70 ℃、540 W 条件下,采用超声波提取仪提取 3 次,每次1 h,合并提取液,减压浓缩得浓缩液。浓缩液上AB-8大孔树脂,选择75%乙醇洗脱液,减压浓缩,真空冷冻干燥得 TFL粉末。亚硝酸钠-硝酸铝-氢氧化钠比色法测得总黄酮质量分数为87.2%。

1.3 试剂

水飞蓟素,纯度≥98%(大连美仑生物技术有限公司,批号:M1120A);CCl4(广州市金华大化学试剂有限公司,批号:171103);丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、IL-6试剂盒(南京建成生物工程研究所,批号分别为20170812,20170927,20170823,20170818,20170924,20170815,20170813,20171108);BCA蛋白浓度测定试剂盒(碧云天生物技术研究所,批号:P0012S);一抗含NLR家族Pyrin域蛋白3(NLRP3)(批号:ab232401)、凋亡相关斑点样蛋白(ASC)(批号:ab47092)、caspase-1(批号:ab179515)、GAPDH(批号:ab181602)及二抗IgG(羊抗兔,批号:ab150077),均为美国Abcam公司。

1.4 仪器

AL104 型电子天平(瑞士梅特勒托利多仪器公司,感量:0.1 mg);RE-52AA 型旋转蒸发仪(上海亚荣生化仪器厂);UV-2501PC 型紫外分光光度计(日本岛津公司)。

1.5 方法

1.5.1 分组及造模 60只昆明小鼠按随机数字表分成6组,正常对照组、模型对照组、水飞蓟素组(水飞蓟素120 mg·kg-1)、TFL小、中、大剂量组(50,100,150 mg·kg-1)。小鼠饲养一周后开始试验,灌胃体积为15 mL·kg-1,正常对照组、模型对照组灌胃等剂量的0.9%氯化钠溶液,每天1次,连续给药7 d。末次给药2 h后,模型对照组及各给药组小鼠按剂量5 mL·kg-1腹腔注射6% CCl4橄榄油溶液,正常对照组小鼠腹腔注射同体积橄榄油,禁食不禁水。

1.5.2 相关指标测定 造模6 h,摘眼球取血,半径为13.5 cm,3000 r·min-1离心 10 min,分离血清,测定血清ALT、AST水平。小鼠处死,取肝脏,4 ℃0.9%氯化钠溶液冲洗,滤纸吸干称重,并计算肝指数(肝指数/%=肝质量×100/体质量)。秤取肝左叶100 mg,加入0.9%氯化钠溶液冰浴匀浆,半径为13.5 cm,4 ℃,3500 r·min-1离心10 min,取上清液,测定IL-1β、IL-6、TNF-α、SOD、GSH-Px、MDA水平。

1.5.3 肝组织学观察 取肝右叶用10%甲醛固定,石蜡包埋,常规切片,苏木精-伊红(HE)染色,并进行常规组织学观察。

1.5.4 蛋白水平的测定 采用Western blotting法,常规方法提蛋白质,调整各样本蛋白总量,参照说明书行SDS-PAGE凝胶电泳,转膜,5%脱脂奶粉封闭2 h,一抗4 ℃孵育过夜,孵育二抗,Bio-Rad凝胶成像系统成像拍照,以GAPDH为内参分析待测蛋白相对表达值。

1.6 统计学方法

采用SPSS15.0版软件,计量数据采用均数±标准差($\bar{x}$±s)表示,采用单因素方差分析,两两比较采用LSD-t检验。除特殊说明外,检验水准α=0.05。以P<0.05表示差异有统计学意义。

2 结果
2.1 多穗柯总黄酮对体质量和肝指数的影响

与正常对照组比较,模型对照组小鼠的肝质量、肝指数均明显升高(P<0.01);与模型对照组比较,水飞蓟素组、TFL中、大剂量组肝质量、肝指数均明显降低(P<0.01),而对小鼠体质量无明显影响。见表1。

表1 多穗柯总黄酮对体质量和肝指数的影响
Tab.1 Effect of TFL on body mass and liver index $\bar{x}$±s,n=10
组别 体质量 肝质量 肝指数/
%
g
正常对照组 27.48±1.12 1.25±0.13 4.55±0.14
模型对照组 27.31±1.08 1.70±0.16① 6.22±0.19①
水飞蓟素组 28.02±1.16 1.38±0.12② 4.93±0.16②
TFL
小剂量组 27.64±1.09 1.61±0.15 5.82±0.18
中剂量组 26.97±1.03 1.47±0.14② 5.45±0.17②
大剂量组 27.93±1.15 1.32±0.11② 4.73±0.17②

①compared with normal control group,t=6.903-22.376,P<0.01;②compared with model control group,t=4.362-16.192,P<0.01.

①与正常对照组比较,t=6.903~22.376,P<0.01;②与模型对照组比较,t=4.362~16.192,P<0.01。

表1 多穗柯总黄酮对体质量和肝指数的影响

Tab.1 Effect of TFL on body mass and liver index $\bar{x}$±s,n=10

2.2 多穗柯总黄酮对ALT、AST的影响

与正常对照组比较,模型对照组小鼠ALT、AST活力均明显升高(P<0.01);与模型对照组比较,水飞蓟素组、TFL中、大剂量组ALT、AST活力均明显降低(P<0.01)。见表2。

表2 多穗柯总黄酮对ALT、AST的影响
Tab.2 Effect of TFL on ALT and AST U·L-1,$\bar{x}$±s,n=10
组别 ALT AST
正常对照组 67.13±11.38 84.32±10.29
模型对照组 203.48±21.28① 170.12±15.48①
水飞蓟素组 149.25±16.03② 98.26±13.46②
TFL
小剂量组 196.37±20.27 160.26±14.27
中剂量组 151.27±17.16② 119.42±12.47②
大剂量组 141.46±15.72② 101.27±10.36②

①compared with normal control group,t=14.616-17.868,P<0.01;②compared with model control group,t=6.46-11.689,P<0.01.

①与正常对照组比较,t=14.616~17.868,P<0.01;②与模型对照组比较,t=6.46~11.689,P<0.01。

表2 多穗柯总黄酮对ALT、AST的影响

Tab.2 Effect of TFL on ALT and AST U·L-1,$\bar{x}$±s,n=10

2.3 多穗柯总黄酮对肝组织 IL-1β、IL-6、TNF-α水平的影响

与正常对照组比较,模型对照组小鼠的肝组织 IL-1β、IL-6、TNF-α水平均明显升高(P<0.01);与模型对照组比较,水飞蓟素组、TFL中、高剂量组肝组织 IL-1β、IL-6、TNF-α水平均明显降低(P<0.01)。见表3。

表3 多穗柯总黄酮对肝组织 IL-1β以及血清IL-6、TNF-α水平的影响
Tab.3 Effect of TFL on the IL-1β level in liver tissue and the serum levels of IL-6 and TNF-α pg·mg-1,$\bar{x}$±s,n=10
组别 IL-1β IL-6 TNF-α
正常对照组 8.82±0.58 8.24±0.38 0.66±0.13
模型对照组 19.28±1.26① 17.01±1.67① 1.48±0.58①
水飞蓟素组 11.12±0.26② 10.02±0.42② 0.79±0.12②
TFL
小剂量组 17.38±1.36 13.28±1.58 1.27±0.46
中剂量组 14.18±0.98② 10.48±1.36② 0.81±0.19②
大剂量组 11.26±0.86② 9.06±1.21② 0.69±0.37②

①compared with normal control group,t=4.362-16.192,P<0.01;②compared with model control group,t=3.471-20.057,P<0.01.

①与正常对照组比较,t=4.362~16.192,P<0.01;②与模型对照组比较,t=3.471~20.057,P<0.01。

表3 多穗柯总黄酮对肝组织 IL-1β以及血清IL-6、TNF-α水平的影响

Tab.3 Effect of TFL on the IL-1β level in liver tissue and the serum levels of IL-6 and TNF-α pg·mg-1,$\bar{x}$±s,n=10

2.4 多穗柯总黄酮对肝组织SOD、GSH、MDA水平的影响

与正常对照组比较,模型对照组小鼠的肝组织SOD、GSH水平均明显降低,MDA明显升高(P<0.01);与模型对照组比较,水飞蓟素组、TFL中、大剂量组肝组织SOD、GSH水平均明显升高,MDA明显降低(P<0.05)。见表4。

表4 多穗柯总黄酮对肝组织SOD、GSH、MDA水平的影响
Tab.4 Effect of TFL on the levels of SOD,GSH and MDA in liver tissue $\bar{x}$±s,n=10
组别 SOD/
(U·mg-1)		 GSH/
(mg·g-1)		 MDA/
(nmoL·mg-1)
正常对照组 104.27±4.78 7.26±1.59 1.55±0.45
模型对照组 38.57±2.48① 3.05±0.78① 4.35±0.53①
水飞蓟素组 88.39±4.15② 5.59±0.66② 2.38±0.48②
TFL
小剂量组 53.46±3.58② 3.75±0.72② 3.68±0.49②
中剂量组 75.28±2.47② 5.08±0.76② 3.12±0.53②
大剂量组 82.47±2.67② 5.72±0.42② 2.42±0.47②

①compared with normal control group,t=7.517-38.581,P<0.01;②compared with model control groupt=5.189-32.587,P<0.01.

①与正常对照组比较,t=7.517~38.581,P<0.01;②与模型对照组比较,t=5.189~32.587,P<0.01。

表4 多穗柯总黄酮对肝组织SOD、GSH、MDA水平的影响

Tab.4 Effect of TFL on the levels of SOD,GSH and MDA in liver tissue $\bar{x}$±s,n=10

2.5 肝组织学观察

模型对照组小鼠肝细胞出现空泡样变,部分肝细胞坏死。水飞蓟素组小鼠肝小叶结构基本正常,空泡样变和肝细胞坏死明显减少;与模型对照组比较,不同剂量多穗柯总黄酮均能有效改善肝组织损伤,空泡样变和肝细胞坏死明显减少。见图1。


图1 急性肝损伤小鼠肝组织病理学变化(×200)
A.正常对照组;B.模型对照组;C.水飞蓟素组;D.TFL小剂量组;E.TFL中剂量组;F.TFL大剂量组。

Fig.1 Pathological changes in liver tissue of mice with acute liver injury (×200)
A.normal control group;B.model control group;C.positive control group;D.low-dose TFL group;E.medium-dose TFL group;F.high-dose TFL group.

2.6 蛋白水平的测定

与正常对照组比较,模型对照组小鼠的肝组织NLRP3、ASC、Caspase-1水平均明显升高(P<0.01);与模型对照组比较,水飞蓟素组、TFL小、中、大剂量组的肝组织NLRP3、ASC、Caspase-1明显降低(P<0.01)。见图2,表5。


图2 6组小鼠蛋白水平测定
1.正常对照组;2.模型对照组;3.水飞蓟素组;4.TFL小剂量组;5.TFL中剂量组;6.TFL大剂量组。

Fig.2 Determination on the protein level in six groups of mice
1.normal control group;2.model control group;3.positive control group;4.low-dose TFL group;5.medium-dose TFL group;6.high-dose TFL group.

表5 多穗柯总黄酮对肝组织NLRP3、ASC、Caspase-1水平的影响
Tab.5 Effect of TFL on NLRP3, ASC and Caspase-1 in liver tissue $\bar{x}$±s,n=10
组别 NLRP3 ASC Caspase-1
正常对照组 0.32±0.07 0.31±0.07 0.18±0.05
模型对照组 0.79±0.11① 0.87±0.09① 0.78±0.08①
水飞蓟素组 0.21±0.05② 0.29±0.06② 0.21±0.07②
TFL
小剂量组 0.69±0.06① 0.72±0.08① 0.68±0.05①
中剂量组 0.31±0.08② 0.51±0.06② 0.28±0.05②
大剂量组 0.24±0.04② 0.32±0.06② 0.24±0.06②

①compared with normal control group,t=11.399-15.531,P<0.01;②compared with model control group,t=3.352-16.956,P<0.01.

①与正常对照组比较,t=11.399~15.531,P<0.01;②与模型对照组比较,t=3.352~16.956,P<0.01。

表5 多穗柯总黄酮对肝组织NLRP3、ASC、Caspase-1水平的影响

Tab.5 Effect of TFL on NLRP3, ASC and Caspase-1 in liver tissue $\bar{x}$±s,n=10

3 讨论

肝损伤主要涉及肝病进展,包括炎症、纤维化、肝硬化和肝细胞癌。正常的肝细胞是容易受到食源性毒素、嗜肝病毒的感染和细菌感染。研究显示,CCl4可通过氧化胁迫和脂质过氧化诱导的急性肝损伤[4]。CCl4 代谢时,可生成大量自由基,诱发脂质过氧化以及级联反应,严重时改变细胞膜的通透性,从而触发肝毒性,并出现肝组织损伤,释放部分活性物质进入血液中[5]。本研究中,模型对照组小鼠血清中AST、ALT水平明显高于对照组,结合病理切片观察,显示模型对照组小鼠肝细胞出现空泡样变,部分肝细胞坏死,说明急性肝损伤的动物模型建立成功。本研究中,TFL给药组ALT,AST水平较模型对照组明显降低,结合病理切片观察,显示不同剂量的TFL均能有效改善肝组织损伤,并具有一定的剂量效应,说明TFL对急性肝损伤大鼠具有一定程度的保护作用。

GSH是一种重要的抗氧化剂,可以清除ROS所致肝毒性。SOD负责将超氧阴离子转化为过氧化氢。 MDA是脂质过氧化标记物。当CCl4诱发较高水平自由基时,GSH、SOD将被大量消耗,此时膜结构损伤明显加重,脂质过氧化产物MDA水平显著增加[6]。因此SOD、GSH和MDA水平能反映CCl4诱发的氧化应激所致肝损伤的程度。在这项研究中,TFL给药可显著增加肝脏抗氧化能力,如其特征在于TFL可以减少CCl4所致氧化应激中MDA水平,升高抗氧化剂SOD、GSH水平,抑制肝细胞脂质过氧化,维持膜的正常结构,从而避免肝细胞的损伤,同时也提示TFL可通过抗氧化应激发挥对肝损伤的保护作用。

越来越多的证据表明,坏死、炎症反应在CCl4诱导的肝细胞毒性中起重要作用[7]。其中TNF-α在肝损伤发生过程中扮演着重要的角色,可诱发肝星形细胞分泌、释放多种炎性递质。同时还会活化中性粒细胞,导致超氧阴离子和蛋白酶水平明显增加,从而产生级联反应,并导致肝损伤的发生。而IL-1β主要可以导致组织局部炎症反应,并作用于单核巨噬细胞,诱发IL-6 的合成和分泌,而IL-6的异常升高会进一步导致肝细胞发生免疫病理反应[8]。因此,TNF-α、IL-1β和IL-6能反映肝损伤的程度[8]。本研究中,TFL对CCl4所致急性肝损伤通过下调炎症诱导肝脏炎症细胞因子如TNF-α、IL-1β和IL-6发挥保护作用。

NLRP3 炎性复合体属于NLR家族成员,主要表达于单核细胞、巨噬细胞等免疫细胞,易被活性氧(ROS)等因素活化。而CCl4诱发的氧化应激所致的膜脂过氧化损伤常导致较高水平的ROS的发生[9]。因此。肝损伤发生时,高水平的ROS的释放是激活 NLRP3 的重要途径,是NLRP3激活的近端信号。当NLRP3 受到内外源性因素影响时,其将发生低聚化,并募集 pro-caspase-1 和ASC 共同组建形成 NLRP3 炎性小体,并调节、活化pro-caspase-1 转变成有活性的Caspase-1,在此基础上,通过剪切IL-1β前体,激活其成熟和分泌,从而发挥生物学作用[10]。因此,调控NLRP3 炎性小体也是对肝损伤发挥保护作用的重要机制。本研究中,TFL能显著降低NLRP3、ASC、Caspase-1水平,表明TFL抑制 NLRP3 炎症小体介导的炎症反应是其发挥对急性肝损伤保护作用的重要机制。

总之,TFL对CCl4诱导的肝细胞损伤的保护作用,与减轻氧化应激水平,改善炎症因子的产生,调控ROS/NLRP3/IL-1β信号通路有关。通过阐明TFL保护CCl4诱导的肝细胞损伤机制,可以为临床急性肝损伤的治疗提供参考,同时也为多穗柯的开发提供依据。

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关键词（key words）
多穗柯总黄酮
肝损伤
急性
抗炎
抗氧化
NLRP3 炎性小体
Total flavonoids from
Liver injury
acute
Anti-inflammatory
Anti-oxidation
NLRP3 inflammatory body
作者
刘晓东
温雯静
赵志军
LIU Xiaodong
WEN Wenjing
ZHAO Zhijun